Abstract

In this work, a novel mechanistic model of hop inhibition beyond the proton ionophore action toward (beer spoiling) bacteria was developed. Investigations were performed with model systems using cyclic voltammetry for the determination of redox processes/conditions in connection with growth challenges with hop-sensitive and -resistant Lactobacillus brevis strains in the presence of oxidants. Cyclic voltammetry identified a transmembrane redox reaction of hop compounds at low pH (common in beer) and in the presence of manganese (present in millimolar levels in lactic acid bacteria). The antibacterial action of hop compounds could be extended from the described proton ionophore activity, lowering the intracellular pH, to pronounced redox reactivity, causing cellular oxidative damage. Accordingly, a correlation between the resistance of L. brevis strains to a sole oxidant to their resistance to hop could not be expected and was not detected. However, in connection with our recent study concerning hop ionophore properties and the resistance of hop-sensitive and -tolerant L. brevis strains toward proton ionophores (J. Behr and R. F. Vogel, J. Agric. Food Chem. 57:6074-6081, 2009), we suggest that both ionophore and oxidant resistance are required for survival under hop stress conditions and confirmed this correlation according to the novel mechanistic model. In consequence, the expression of several published hop resistance mechanisms involved in manganese binding/transport and intracellular redox balance, as well as that of proteins involved in oxidative stress under “highly reducing” conditions (cf. anaerobic cultivation and “antioxidative” hop compounds in the growth medium), is now comprehensible. Accordingly, hop resistance as a multifactorial dynamic property at least implies distinct resistance levels against two different mechanisms of hop inhibition, namely, proton ionophore-induced and oxidative stress-induced mechanisms. Beyond this specific model of hop inhibition, these investigations provide general insight on the role of electrophysiology and ion homeostasis in bacterial stress responses to membrane-active drugs.

The inflorescences of the hop plant Humulus lupulus are traditionally used for beer brewing, due to the pleasant bitterness of hop compounds in beer and their additional inhibitory effect on bacteria. These antibiotic and bacteriostatic properties of hops comprise several inhibitory mechanisms. The described effects of hop compounds on bacteria are permeability changes in the bacterial cell wall (30), leakage of the cytoplasmic membrane and a subsequent inhibition of respiration, and protein, DNA, and RNA synthesis (42), as well as changes in leucine uptake and proton ionophore activity (33). In a recent study (5), we characterized the latter ionophore properties of hop compounds in a cell-free model system via bilayer lipid membrane (BLM) techniques in connection with growth challenges of hop-sensitive and -resistant Lactobacillus brevis strains. The antibacterial action of iso-α-acids as proton ionophores could be confirmed by the BLM measurements; however, that the reported ionophore properties enable electroneutral H+/Mn2+ exchange (34) could not be verified. On the other hand, the measurements indicated the manganese-dependent enhancement of transmembrane charge permeation. The origin of high membrane potential formation in the presence of manganese, as well as strongly elevated membrane conductivity with a concomitant increase in the effectiveness of an uncoupler, suggests a different origin of charge transfer under these conditions. Accordingly, the expected cross-resistance to sole proton ionophores could not be detected (5), and any antibiotic tested until now could not simulate the stress induced by hop compounds in bacteria (11). The latter observation may be related to the complexity of the chemical composition of hops, which is due to compound variations resulting from variations in the natural product as well as to consecutive chemical conversions of hop compounds, which are known to be highly reactive. This high level of reactivity of hop compounds comprises polymerization as well as reduction and oxidation processes (6, 36). The latter are also known to efficiently inhibit bacteria by causing oxidative stress, in which oxidizing molecules are generated at a higher rate than they are detoxified. To counteract oxidative stress (7, 12, 20, 21, 41, 44, 46), bacteria developed several defense strategies, namely, reduced generation of oxidizing molecules during metabolism, enzymatic or nonenzymatic detoxification of the latter, and repair of damaged cell components (20). In this context, the importance of the nonenzymatic detoxification mechanism of oxidative molecules in lactic acid bacteria is connected to their extraordinary high levels of intracellular manganese (1). Mn2+ can cycle in vivo between the oxidation states +2 and +3, whereas the ligand environment alters the redox potential. Thus, “activated” Mn2+ can act as a scavenger of toxic oxygen species (17), whereas Mn3+ and its complexes are able to oxidize different substrates and contribute to the overall oxidative potential (24). However, in the presence of hop compounds, hop-resistant L. brevis TMW 1.465 strongly reduced intracellular manganese levels (normally elevated under environmental stress conditions [17]) and changed the protein expression profile, leading to an elevated rate of manganese binding and/or manganese-dependent enzymes as well as to the expression of repair proteins and proteins of membrane lipid synthesis, which counteract oxidative damage (3, 4). This unusual regulation could possibly be linked to the fact that the ligand environment (e.g., iso-α-acids [34]) alters the redox properties of manganese. On the other hand, the possibility cannot be excluded that the redox properties of hop compounds, which are known to be highly redox-reactive substances (6), are altered in the presence of manganese. Accordingly, a study of the redox reactivity of hop compounds in connection with divalent manganese is required.

An appropriate method to investigate possible oxidation processes induced by hop compounds and their manganese complexes (34, 37) is cyclic voltammetry (voltamperometry). This method can be applied in aqueous solutions (28, 39) as well as upon reconstituted planar bilayer lipid membranes (14-16, 29). The use of cyclic voltammetry for determination of redox processes/conditions as described for hop compounds containing beer samples has considerable advantages compared to potentiometric measurements, as it is fast and reproducible (39). Further, cyclic voltammetry is an elegant tool for the study of transmembrane redox reactions in electron-conducting BLMs. These transmembrane redox processes play a central role in life's membrane bioenergetics (43). The possibility of the application of cyclic voltammetry to a BLM resides in the double-electrode behavior of the planar membrane (8). The electron transport process through the BLM implies electron transfers at both solution-membrane interfaces, as well as an electron transfer through the BLM. Consequently, redox couples taking part in the process must be located at the membrane-solution interfaces as well as inside the membrane in their oxidized and reduced form. These redox couples are not necessarily the same in solutions and membranes. Nevertheless, for transmembrane electron transfer, proximity of the redox potentials of both is required (29). As lactic acid bacteria can contain millimolar concentrations of membrane-impermeable divalent manganese located near the cytoplasmic membrane (1) and hop compounds are amphiphilic compounds, a great deal of the above-described assumptions required for transmembrane redox processes is fulfilled. Consequently, redox characteristics of membrane-active hop-derived substances were investigated, the results of which can undoubtedly contribute to the understanding of biological membrane processes under hop stress conditions (14).

MATERIALS AND METHODS

Cyclic voltammetry and the electrochemical indicator time test (ITT) (39) were used to assess the redox properties of hop compounds (Isohop precipitated and dissolved in methanol [5]; Johann Barth & Sohn GmbH & Co., Nürnberg, Germany) at various pH values with and without the addition of MnCl2. Iso-α-acids (the isoextract contained 98.4% [wt/vol] iso-α-acids [5]; thus, the terms hop compounds and iso-α-acids are subsequently used as synonyms) were dissolved at concentrations of 100 μM in buffer (50 mM acetic acid, 50 mM potassium phosphate, 5 mM CAPS [N-cyclohexyl-3-aminopropanesulfonic acid], and 0.5 g liter−1 KCl). pH values ranged from 4 to 11 in steps of 1 pH unit. Where indicated, MnCl2 was added at concentrations of 200 μM. All buffers and samples were degassed and stored under nitrogen. The redox analysis was performed with a modified APP 5003 analytic automaton instrument (ME Instrument, Trappenkamp, Germany) as previously described (39). In short, cyclic voltammetry was performed with a starting potential of 0.0 V and a switching potential of 1.1 V versus that of the normal hydrogen electrode ([NHE] scan rate, 0.4 V s−1). The zero current potentials for forward and reverse scans were determined, and the mean zero current potentials were calculated. The ITT was measured with a working potential of 0.625 V versus that of the NHE, and the 2,6-dichlorophenol-indophenol (DCI) reaction time was set to 5 min. Reagent solutions of 0.005 M DCI and 0.001 M ascorbic acid and an equimolar amount of oxalic acid were used (dosage, 0.55 ml per 8.80 ml of sample). The reductone-ascorbic acid ratio was calculated as previously described (39).

Cyclic voltammetry measurements of bilayer lipid membranes in the presence of isomerized hop compounds were performed with a custom-made microchip-controlled two- or four-electrode capacitance measurement and potentiostat/galvanostat system (15, 16), principally as previously described (5). For cyclic voltammetry experiments, the potential at the BLM was scanned between ±100 mV at a scan rate of 0.3 mV s−1 (39, 43). During the scan, the current passing through the working electrode (BLM) was measured and plotted against the applied voltage. The measurements were performed thermostatically (with a MultiTemp III instrument; Amersham Biosciences Europe GmbH, Freiburg, Germany) at 23°C. Membrane lipid egg phosphatidylcholine (Lipoid E PC S; Lipoid GmbH, Ludwigshafen, Germany) was used (5). The membrane-forming mixture contained 20 mg phosphatidylcholine (PC) and 20 mg cholesterol (22) per 1 ml decane. Isomerized hop extract (Isohop precipitated and dissolved in methanol [5]; Johann Barth & Sohn GmbH & Co., Nürnberg, Germany), membrane-impermeable electron donors and acceptors (K3[Fe(CN)6]/K4[Fe(CN)6] or ascorbic acid/K3[Fe(CN)6]/K4[Fe(CN)6]), and MnCl2 as the salt of the redox-reactive divalent cation were used. Unless otherwise stated, KCl (0.2 M) was used as a supporting electrolyte. Electrolyte solutions were buffered with Tris (25), MES [2-(N-morpholino)ethanesulfonic) acid], citric acid (5 mM), or potassium phosphate (20 mM). All solutions were stored under nitrogen.

Correlation of hop action in BLMs to hop resistance in bacteria.

Cultures were grown to stationary phase (the final optical density at 590 nm [ODf590 nm] is indicated in brackets below) at 30°C in mMRS4 medium (40) and washed twice (for 10 min each at 5,000 × g) with assay medium without oxidants. Growth challenges with hop-sensitive L. brevis TMW 1.6 (ODf590 nm = 3.5) and 1.1369 (ODf590 nm = 4.6) and hop-resistant TMW 1.313 (ODf590 nm = 3.6) and 1.465 (ODf590 nm = 3.6) were done in mMRS4 medium without cysteine at pH 4.3 (the divalent cation content of mMRS4 was reduced to 98 mg/liter magnesium and 0.16 mg/liter manganese [5]) and with additions of various concentrations of oxidants: H2O2 (maximum concentration tested [Cmax] = 5.0 mM), peroxyacetic acid ([PES] Cmax = 0.5 mM), diamide (Cmax = 2.0 M), and paraquat (Cmax = 0.2 M). The concentrations of oxidants were varied in 10% steps from 0 to 100% of Cmax. Experiments with controls were performed without the addition of oxidants. Growth challenges were carried out in microtiter plates sealed with parafilm, using resazurin as a metabolic growth indicator (27). The inoculation density was set to an OD590 nm of 0.10. The metabolic tests were analyzed after a 3-day incubation at 30°C via the addition of 80 mM Tris-HCl (pH 8.8) to each well in order to achieve a pH in the range of 5.5 to 11.0, where resazurin appears blue in oxidized form and pink in its reduced form, resorufin (5).

RESULTS

To determine the redox properties of hop compounds in aqueous solutions with various pH values and the presence of MnCl2, automated cyclic voltammetry and electrochemical ITT analysis were performed. The mean zero current potentials ([MZP] cyclic voltammetry) and the reductone-ascorbic acid ratios (electrochemical ITT) of hop compounds at various pH values were determined. The cyclic voltammograms from hop samples (data not shown) showed cyclic voltammogram shapes typical for beer. The calculated MZP is assumed to deliver some information about the redox condition of the sample, where high MZP are associated with a more oxidizing condition and low MZP with the opposite (39). Figure ​Figure11 depicts the MZP values of 100 μM iso-α-acids in buffer and 100 μM iso-α-acids plus 200 μM MnCl2 in buffer. As controls, buffer alone and buffer with 200 μM MnCl2 were used. In general, a decrease in MZP with rising pH values was obtained for all samples. A comparison of iso-α-acids in the presence of MnCl2 and pure iso-α-acids shows that MZP values in the presence of MnCl2 are mainly below those found for pure iso-α-acids (except at pH 6 and pH 11), indicating that the iso-α-acids in the presence of MnCl2 represent the more reducing condition (ΔMZPat pH value = MZPbuffer and iso-α-acids − MZPbuffer and iso-α-acids plus MnCl2, the ΔMZP at different pH values were as follows: ΔMZPpH 4 = 7.8 ± 0.3 mV; ΔMZPpH 5 = 11.2 ± 1.9 mV; ΔMZPpH 7 = 8.0 ± 5.6 mV; ΔMZPpH 8 = 15.0 ± 10.6 mV; ΔMZPpH 9 = 31.8 ± 5.8 mV; and ΔMZPpH 10 = 35.4 ± 3.5). The same samples used for cyclic voltammetry were analyzed via electrochemical ITT. In this measurement, the reductone level of the sample was determined and standardized with a known amount of ascorbic acid as a reducing agent. Thus, a high reductone-ascorbic acid ratio is correlated with a more reducing condition of the sample. No significant differences in the reductone level of iso-α-acids in the presence or absence of MnCl2 were obtained in the pH range from 4 to 9 (reductone level, 0.09). Beyond pH 9, an increase in reducing power for iso-α-acids as well as iso-α-acids in the presence of MnCl2 was monitored (maximum reductone level at pH 11, 0.8).

Cyclic voltammetry data from BLMs in the presence of hop compounds.

Cyclic voltammetry data of BLMs were recorded in the presence of the hop compounds with various pH values and MnCl2 concentrations and in the presence of membrane-impermeable electron donors and acceptors in aqueous solutions. According to Sobiech et al. and Tien (39, 43), voltammograms were recorded at a slow scan rate of 0.3 mV s−1 in the range of −100 mV to 100 mV, since the reaction times of beer-associated redox couples (e.g., hop polyphenols and hop bitter acids) cover the range from 15 s to 150 min. The breakdown voltage of the BLMs limits the switching potential. For all conditions, three subsequent scans were run. No separate peaks could be identified under all conditions tested (39). Cyclic voltammograms recorded at various pH values, with or without the addition of MnCl2 or membrane-impermeable electron donors and acceptors but without hop compounds in the aqueous solution, coincided with the x axis on the scale used. Cyclic voltammograms of BLMs at pH values of 4.0, 7.0, and 10.0 (virtually identical in shape to the cyclic voltammogram at pH 7; data not shown) were recorded in the presence of hop compounds (Fig. ​(Fig.2).2). The hop compound concentrations were obtained from conductivity experiments (25 mV potential; data not shown) in order to achieve comparable membrane conductance independently of pH value. The cyclic voltammograms for pH values of 7.0 and 10.0 were symmetric toward the origin and show the typical shape of cyclic voltammograms of ionophores (e.g., valinomycin) (31). The forward and backward scans virtually coincide. The rise in conductivity in subsequent potential sweeps can be attributed to an increase in hop compound concentrations in the membrane over time. In contrast, the cyclic voltammogram from the pH 4.0 condition was highly asymmetric toward the origin. The anodic current through the BLM (cycle 1) at 100 mV of applied potential was more than 10 times higher than that obtained at pH 7.0 and more than 20 times higher than that at pH 10.0 (0.10 nA). For the cathodic current at the opposite end of the potential sweep, a nearly triple value (with regard to pH 7.0) and a fivefold value (at pH 10.0; 0.18 nA) were obtained. The positive forward scan at pH 4.0 led to a significantly higher current through the BLM than the backward scan. In contrast to the cyclic voltammograms recorded at pH 7.0 and pH 10.0, the conductivity decreased in subsequent scans. To prove that a transmembrane redox reaction was responsible for the highly asymmetric current/voltage (I/U) curve at low pH, membrane-impermeable electron donors and acceptors (K3[Fe(CN)6]/K4[Fe(CN)6] or ascorbic acid/K3[Fe(CN)6]/K4[Fe(CN)6]) were added to the aqueous solutions on opposite sides of the membrane. As the choice of water-soluble redox couple did not alter the measurement result, only data for the first redox couple are shown. It was ascertained that the iron part of the redox couples did not act as a di- or trivalent cation, which would form a complex with the hop compounds {Fe2+ and Fe3+ hop compound complexes are intensely orange, which distinguishes them from the yellow-green of K3[Fe(CN)6] and K4[Fe(CN)6]}. A comparison of cyclic voltammograms recorded with or without the addition of K3[Fe(CN)6]/K4[Fe(CN)6] is depicted in Fig. ​Fig.3.3. The anodic current increased more than 2.5-fold with the presence of an electron donor and acceptor couple in the aqueous solutions, and the cathodic current increased more than 7-fold. The elevated cathodic current can be attributed to the redox couples formed during the positive scan, which can undergo reversible reactions. When the redox couple in the aqueous solution was added in inverse compartments of the electrolytic cell {positive scan, K4[Fe(CN)6] at the reducing side of the BLM and K3[Fe(CN)6] at the oxidizing side of the BLM}, the I/U curve of the scan was nearly identical to those recorded without the addition of the redox couple. The influence of MnCl2 additions in both or one compartment of the electrolytic cell was recorded. The presence of MnCl2 on both sides of the membrane resulted in a curve similar to that in the presence of the K3[Fe(CN)6]/K4[Fe(CN)6] redox couple but with less markedness. The anodic current increased more than 2.5-fold with the presence of MnCl2; the cathodic current increased more than 3-fold. Figure ​Figure44 shows a comparison of cycle 3 data for different modes of addition of MnCl2 with cycle 3 data for the addition of electron donors and acceptors. Cycles 3 were chosen for the comparison because mainly reversible reactions (checked by running six additional cycles that exhibited similar shapes), which are much easier to evaluate, were taking place. A comparison of data for the mode of MnCl2 addition with data for the addition of K3[Fe(CN)6]/K4[Fe(CN)6] to the aqueous solutions for cycle 3 shows that the Mn2+ hop compound complex can act as an electron donor. It is obvious that the two voltammograms are virtually identical. For example, in the positive scan with MnCl2 added at the oxidizing side of the BLM, the current rose compared to that recorded without the addition. The current in the negative cycle was not altered under this condition. The same is true when the electron donor was added at the trans side (oxidizing in the positive voltage sweep) of the electrolytic cell and the electron acceptor on the opposite side. The offset measured at zero voltage for the voltammograms with the addition of MnCl2 is attributed to the potential generated by a difference in MnCl2 concentrations of the solutions separated by the membrane (5).

Cyclic voltammograms for charge transfer through the BLM (PC) in the presence of iso-α-acids in both compartments of the electrolytic cell at the indicated pH. Three subsequent scans are displayed. The scan rate was 0.3 mV s−1. The aqueous...

Cyclic voltammograms for charge transfer through the BLM (PC) in the presence of 10 μM iso-α-acids in both compartments of the electrolytic cell with (right panel) or without (left panel) the addition of 0.5 mM K3[Fe(CN)6] (cis side) and...

Cyclic voltammograms for charge transfer through the BLM (PC) in the presence of 10 μM iso-α-acids in both compartments of the electrolytic cell dependent on the mode of addition of 25 μM MnCl2 (right panel) or 0.5 mM K3[Fe(CN)...

Correlation of hop action in BLMs to hop resistance in bacteria.

Growth challenges with hop-sensitive L. brevis TMW 1.6 and 1.1369 and hop-resistant TMW 1.313 and TMW 1.465 were done in the presence of the oxidants H2O2, PES (intracellular H2O2 forming), paraquat (O2 forming), or diamide (SH group oxidizing). The MICs are given in Table ​Table1.1. The levels of resistance of hop-sensitive strains to H2O2 or O2-forming oxidants varied strongly, while they differed less with regard to SH group-oxidizing diamide. Comparison of the levels of resistance to oxidants of hop-sensitive and -resistant L. brevis strains indicated high and low levels of resistance for hop-sensitive L. brevis strains TMW 1.6 and TMW 1.1369, respectively, while the levels of resistance for both hop-resistant strains TMW 1.313 and TMW 1.465 were at medium levels and did not unambiguously correspond to hop resistance.

MICs of hop compounds and different oxidants for hop-sensitive L. brevis strains TMW 1.6 and 1.1369 and -resistant strains TMW 1.465 and 1.313a

DISCUSSION

Mode of action of hop compounds in model systems.

The mode of action of hop compounds as ionophores in bilayer lipid membranes was characterized in a recent study (5) and indicated, beyond the hop action as proton ionophores, a manganese-dependent enhancement of transmembrane charge permeation that could not be attributed to the described electroneutral H+/Mn2+ exchange mechanism (34). In order to clarify the interrelation of hop compound actions, the presence of MnCl2, and the solution pH with regard to possible redox properties of manganese and/or hop compounds, cyclic voltammetry was used as an analytical tool for the determination of redox reactions. In this context, hop compounds, as well as manganese, exhibit distinct redox properties (6), which vary according to environmental parameters. It is known that the ligand environment (e.g., bicarbonate or possibly iso-α-acids) alters the redox properties of manganese (17). On the other hand, the possibility that the redox properties of hop compounds are altered in the presence of manganese cannot be excluded. In association with this, the pH dependence of redox potential must be mentioned. For manganese, which has as complicated a redox chemistry as any element known, this pH dependence can be obtained from the Frost diagrams (free energies versus oxidation state) constructed for acidic and basic solutions. If we focus on Mn(II) and Mn(III), the Frost diagrams show a thermodynamic well for Mn(II) in acidic solutions and for Mn(III) in basic solutions. Thus, free Mn(II) can act as a reducing agent in basic solutions, while the tendency to react in acidic solutions is low. On the other hand, Mn(III) acts as a potent oxidizing agent at acidic pH (32). In the same manner, the redox properties of iso-α-acids could be altered by pH as well. To support the above-stated considerations, the redox properties of hop compounds were initially investigated in aqueous solutions (without membrane participation) with regard to pH value and the presence of manganese via automated cyclic voltammetry and electrochemical ITT. The cyclic voltammetry data confirmed the influence of Mn2+ on the redox properties of iso-α-acids. Measurements suggest an elevated reduction power of hop compounds in the presence of MnCl2. Further, an increase in oxidation power toward low pH values was monitored, with a higher degree of markedness for pure iso-α-acids in comparison to iso-α-acids in the presence of MnCl2. However, the differences between controls and samples were small in this case. This can be attributed to the low iso-α-acid concentrations in the samples (100 μM), which could not be elevated due to the poor solubility of iso-α-acids in aqueous solutions at low pH in the presence of divalent cations. The ITT data showed that the reductone level of hop compounds was not altered by the presence of MnCl2, indicating that the reducing power of hop-derived enediol compounds was not affected by MnCl2. In conclusion, the cyclic voltammetry data support the suggestion that differences in pH and/or MnCl2 concentrations upon a membrane can drive transmembrane redox reactions mediated by hop compounds.

In order to confirm or disprove this hypothesis and consider the influence of membrane participation, cyclic voltammetry for detection of redox reactions in BLMs (43) was used. Such redox-driven transmembrane charge transfer is, e.g., described for electron conducting TCNQ (7,7′,8,8′-tetracyano-p-quinodimethane)-doped BLMs in the presence of appropriate redox couples on opposite sides of a membrane (43). As an initial experiment, the pH dependence of cyclic voltammograms of iso-α-acids on both sides of the BLM was recorded (Fig. ​(Fig.2).2). At higher pH values (pH 7. 0 and pH 10.0), the cyclic voltammograms showed a shape that is typical for ionophores (e.g., valinomycin) (5, 31). At a low pH of 4.0, this behavior completely changed. The cyclic voltammogram was highly asymmetric toward the origin, the forward and backward scans differed significantly, and high currents through the membrane were recorded. In contrast to the cyclic voltammograms recorded at high pH, the conductivity of the BLM in the pH 4.0 condition decreased in subsequent scans. If redox reactions were taking place, the latter can be attributed to the fact that most redox-reactive organic compounds undergo irreversible reactions (39). The asymmetry and shape of the cyclic voltammograms and the decreased conductance in subsequent potential sweeps recorded at low pH leave little doubt that transmembrane redox reactions were taking place, with electrons moving through the BLM from one membrane-solution interface to the other. In this case, the electron transport through the BLM requires both oxidized and reduced forms of the redox couple in the BLM as well as redox couples in the aqueous phase providing and accepting electrons at the membrane-solution interfaces (29). Accordingly, the supply of additional membrane-insoluble redox couples in the aqueous solutions enhances the electron transfer through the BLM, as monitored by the addition of K3[Fe(CN)6] and K4[Fe(CN)6] on opposite sides of the BLM in the presence of iso-α-acids (Fig. ​(Fig.3).3). To get insight into the mechanisms of charge transfer mediated by hop compounds in the presence of manganese gradients and their influence on transmembrane redox reactions, cyclic voltammograms were compared to those recorded in the presence of electron donors and acceptors (Fig. ​(Fig.4).4). It was demonstrated that the Mn2+-hop compound complexes can act as electron donors, while the pure iso-α-acids can mediate the acceptor part. These data are consistent with the measurements of redox potential of hop compounds with and without MnCl2 by automated cyclic voltammetry without membrane participation, which determined the pure iso-α-acids as the more oxidizing condition (capable of taking up electrons) and the manganese-hop complexes as the more reducing condition (providing those electrons). Thus, the mode of antibacterial action of hop compounds can be extended from a proton ionophore to a redox-reactive uncoupler.

Relevance of this study to understanding the antibacterial action of hop compounds.

Antibacterial hop compounds, mainly iso-α-acids, are described as ionophores, which transport H+ into the bacterial cells and thus dissipate ion gradients across the cytoplasmic membrane (5, 26, 31, 34). In several studies, it was demonstrated that the main factors affecting the antibacterial activity of hop compounds are the pH value and their ability to bind to cations such as Mn2+ (34, 35, 38). Monovalent cations stimulated the proton ionophore action of hop compounds (37, 38). However, the therefore-expected cross-resistance to other proton ionophores (e.g., carbonyl cyanide m-chlorophenylhydrazone [CCCP]) could not be detected (11). In a recent study, the mode of action of iso-α-acids as proton ionophores was confirmed by BLM measurements (5). However, the proton ionophore action was not restricted to low pH values and was distinct up to pH 9 and higher pH values. These findings are not in contradiction to the results of Simpson and colleagues (33-38), since it is known that the cellular proton gradient is dispensable at pH values in the range of the normal intracellular pH of the bacterium (about 6.5 for L. brevis) if other ion gradients such as potassium or sodium, for example, take over its function as a driving force (23). In this context, the role of extracellular monovalent cations, beyond their cooperative binding to hops (37), could reside in the change in ion gradients upon the bacterial membrane needed for survival under low proton gradients. The change from the proton gradient to, e.g., sodium or potassium gradients as the driving force under hop stress is supported by the fact that the membrane potential (ΔΨ) in the presence of hop compounds (34) was altered to a lesser extent in comparison to the proton gradient.

The role of manganese in this antibacterial mechanism, which was found to strongly enhance the transmembrane charge permeation in model systems (5), is described subsequently. As it is known that both manganese and hop compounds are highly redox-reactive substances (6, 32), this fact was initially taken into consideration. With evidence from multiple experiments, it could be demonstrated that Mn2+-hop compound complexes, as well as the hop compound itself, take part in transmembrane redox reactions. Such redox reaction-based agents are known to be highly efficient in the μM range (19). Thus, it can be understood why changes as small as 0.2 in extracellular pH driving the proton ionophore action as well as shifting the redox potentials can result in a 50% altered antibacterial effect of hop compounds (34). Unfortunately, transmembrane and intracellular redox reactions are hard to monitor in vivo. This is due to the fact that the permeability of the cells for fluorescence-based detectors of intracellular oxidative stress, like hydroxy-phenyl fluorescein, can be influenced by the addition of a stress-causing antibiotic. Accordingly, it is proposed that a more reliable approach for the detection of intracellular oxidative stress is the measurement of the expression levels of associated stress proteins in the bacterium (13). These oxidative stress-associated proteins (9, 18, 44) were recognized in hop-stressed L. brevis via proteomics as cyclopropane-fatty acyl-phospholipid synthase (CFA), formamidopyrimidine-DNA glycosylase, HitA, and several oxidoreductases (3), indicating that transmembrane redox reactions mediated by hop compounds and their manganese complexes cause intracellular oxidative stress. A proposal for the extended antibacterial mechanism of hop compounds is delineated subsequently.

Inside the bacteria, a higher pH (normally about 0.5 to 1 pH unit) with respect to the outside pH and high concentrations of Mn2+ are present (1). As hop compounds penetrate the cytoplasmic membrane, they come in contact with intracellular Mn2+ and form complexes (37) (higher pH and Mn2+ concentrations = a reducing condition). Thus, an electron donor is formed at the inner membrane-solution interface. The electrons are transferred through the membrane to the iso-α-acids at the opposite membrane-solution interface, which act as electron acceptors (lower pH and Mn2+ concentrations = an oxidizing condition). As irreversible as well as reversible redox reactions were identified by BLM experiments, the remaining oxidized manganese-hop compound complexes inside the bacterium could now act as electron acceptors, causing oxidative stress. It is known that mainly metal ion redox couples such as Mn(II)/Mn(III) undergo reversible redox reactions (39). Thus, if we consider the manganese part of the manganese-hop compound complex as a possible electron donor/acceptor and remember that there is a thermodynamic well for Mn(II) at lower pH values, the driving force for intracellular oxidation takes shape. The preference for directing the reducing power of the Mn2+-hop compound complexes through the membrane to the outside can reside in the proximity between the redox potentials of the redox species in the membrane and those in the aqueous phase, which are stringently required for such reactions (29). In conclusion, the mode of antibacterial action of hop compounds is suggested to consist of mechanisms of proton ionophore (5) and redox-reactive uncoupler activities occurring in parallel.

Accordingly, correlation of a sole-oxidant resistance in L. brevis strains to hop resistance could not be expected and was not detected (Table ​(Table1).1). In this connection, the use of resazurin as a metabolic growth indicator enabled fast and distinct detection of bacterial survival (27). However, the range of its application is restricted to pH values of 5.5 to 11.0 for the detection/growth medium. Below pH 5.5, both resazurin (oxidized form) and resorufin (reduced form) appear pink. Accordingly, an upshift in pH after incubation (5) was used for discrimination of cell metabolic activity (pink color of reduced-form resorufin) and cell death (blue color of the oxidized form of resazurin [27]). Considering the above-suggested mechanisms of hop inhibition, the detected differences in resistance levels toward oxidants (Table ​(Table1)1) in connection with the resistance levels toward ionophores (5) enable a clear discrimination of hop-sensitive and -resistant L. brevis strains on the basis of hop compound action-comprising stresses (Table ​(Table2).2). We suggest that both ionophore and oxidant resistance is required to survive the stress caused by hop compounds (at a low pH value, as found in beer) according to the novel mechanistic model. This is in good correlation with the results obtained from growth challenges of hop-sensitive and -resistant L. brevis strains in the presence of proton ionophores as well as under oxidative stress conditions. As shown in Table ​Table2,2, hop-sensitive L. brevis TMW 1.6 exhibits high and low levels of resistance to oxidative stress and proton ionophores, respectively, while the opposite is true for hop-sensitive L. brevis strain TMW 1.1369. Thus, both strains miss one of the suggested resistance levels required to survive the hop stress condition. In contrast, the hop-resistant strains exhibit a high level of ionophore resistance and at least a medium level of resistance (compared to highly oxidant-resistant strain L. brevis TMW 1.6) against oxidative stress. Furthermore, a proteomic study (3) and investigations of cellular manganese levels in L. brevis TMW 1.465 (4) indicate that the unusual downregulation of intracellular manganese levels under environmental stress conditions in this case effectively counteracts the manganese-dependent inhibitory transmembrane redox reaction of hop compounds. In parallel, the upregulation of manganese binding/dependent enzyme levels compensates for lost enzyme activity, and the expression of repair proteins and proteins, which counteract oxidative damage, ensures survival under hop stress conditions. In consequence, the expression of several hop resistance proteins involved in manganese binding and intracellular redox balance, as well as proteins of oxidative stress (3) under “highly reducing” conditions (cf. anaerobic cultivation and “antioxidative” hop compounds [10, 45] in the growth medium), is now comprehensible. Accordingly, hop resistance as a multifactorial dynamic property (2, 3) at least implies distinct resistance levels against two different mechanisms of hop inhibition, namely, proton ionophore-induced and oxidative stress mechanisms. Beyond the specific model of hop inhibition, these investigations provide general insight on the role of electrophysiology and ion homeostasis in bacterial stress responses to membrane-active drugs.